Process for the fermentative preparation of D-pantothenic acid and/or its salts

ABSTRACT

A method for the fermentative preparation of D-pantothenic acid and/or its salts or feedstuff additives containing these. Said method comprising, fermentation of Escherichia coli microorganisms wherein the nucleotide sequence(s) coding the glyA gene is overexpressed.

This application claims the benefit of priority to Germany 101 06 461.6filed Feb. 13, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for the fermentativepreparation of D-pantothenic acid and/or its salts or mixturescontaining these using microorganisms from the Enterobacteriaceaefamily, in which at least the glyA gene is enhanced.

2. Description of the Background

Pantothenic acid is produced all over the world in amounts of severalthousand tons per year. It is used, inter alia, in human medicine, inthe pharmaceutical industry and in the foodstuffs industry. A highproportion of the pantothenic acid produced is used for feedingeconomically useful animals such as poultry, and pigs. The demand forthis material is increasing.

Pantothenic acid can be prepared by chemical synthesis or biotechnicallyby the fermentation of suitable microorganisms in suitable nutrientmedia. In the case of chemical synthesis, DL-pantolactone is animportant precursor. This is prepared in a multi-step process fromformaldehyde, isobutylaldehyde and cyanide, the racemic mixture isresolved in a subsequent process step, D-pantolactone is condensed withβ-alanine and D-pantothenic acid is obtained in this way.

The typical commercial form is the calcium salt of D-pantothenic acid.The calcium salt of the racemic mixture D,L-pantothenic acid is alsocommonly available.

The advantage of fermentative preparation by microorganisms is thedirect formation of the desired stereoisomeric form, that is the D-form,which contains no L-pantothenic acid.

Various species of bacteria such as, e.g. Escherichia coli (E. coli),Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacteriumammoniagenes and also yeasts, such as e.g. Debaromyces castellii can, asshown in EP-A 0 493 060, produce D-pantothenic acid in a nutrient mediumwhich contains glucose, DL-pantoic acid and β-alanine. Furthermore, EP-A0 493 060 shows that, in the case of E. coli, the formation ofD-pantothenic acid is improved by the amplification of pantothenic acidbiosynthesis genes from E. coli which are contained on the plasmids pFV3and pFV5, in a nutrient medium which contains glucose, DL-pantoic acidand β-alanine.

EP-A 0 590 857 and U.S. Pat. No. 5,518,906 describe mutants derived fromE. coli strain IF03547, such as FV5714, FV525, FV814, FV521, FV221,FV6051 and FV5069 which carry resistance to various antimetabolites suchas salicylic acid, a-ketobutyric acid, β-hydroxyaspartic acid,O-methylthreonine and a-ketoisovaleric acid. They produce pantoic acidin a nutrient medium which contains glucose, and D-pantothenic acid in aglucose and B-alanine-containing nutrient medium. Furthermore, in EP-A 0590 857 and U.S. Pat. No. 5,518,906, it is stated that the production ofD-pantoic acid is improved in a glucose-containing nutrient media andthe production of D-pantothenic acid is improved in a nutrient mediumwhich contains glucose and β-alanine after amplification, in the strainsmentioned above, of the pantothenic acid biosynthesis genes panB, panCand panD, which should be present on the plasmid pFV31.

Furthermore, WO 97/10340 reports on the beneficial effect of enhancingthe ilvGM operon on the production of D-pantothenic acid. Finally,EP-A-1001027 reports on the effect of enhancing the panE gene on theformation of D-pantothenic acid. According to known procedures,D-pantothenic acid or the corresponding salt is isolated from thefermentation broth and purified (EP-A-0590857 and WO 96/33283) and thenused in purified form or the entire D-pantothenic acid-containing brothis dried EP-A-1050219) and used in particular as a foodstuffs additive.

In view of the increasing demand for D-panthothenic acid, there remainsa need for new methods of producing this material.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods of makingD-pantothenic acid and salts thereof, as well as feedstuffs additivescontaining the same.

The invention provides a process for the preparation of D-pantothenicacid and/or its salts or foodstuffs additives which contain, in additionto these, further constituents from the fermentation by fermentation ofmicroorganisms from the Enterobactericeae family, in particular thosewhich already produce D-pantothenic acid, in which

(a) the nucleotide sequence(s) in the microorganisms coding for theendogenous glyA gene is enhanced, in particular overexpressed, underconditions which are suitable for the production of serine hydroxymethyltransferase,

(b) D-pantothenic acid and/or its salts are enriched in the medium or inthe cells of the microorganisms and

(c) the desired products are isolated after completion of fermentation,wherein an amount of ≧0 to 100% of the biomass and/or optionally furtherconstituents of the fermentation broth are separated,

wherein the microorganisms produce D-pantothenic acid.

The invention also provides a process in which, after completion offermentation, all or some of the biomass remains in the fermentationbroth and the broth obtained in this way is processed, optionally afterbeing concentrated, to give a solid mixture which contains D-pantothenicacid and/or its salts and which also contains other constituents of thefermentation broth.

Thus, the present invention provides a method of producing D-pantothenicacid and/or a salt thereof, comprising:

fermenting a microorganism of the family Enterobacteriaceae, in whichthe nucleotide sequence for the endogenous glyA gene is enhanced, in amedium suitable for the production of serine hydroxymethyl transferase,wherein the microorganism produces the D-pantothenic acid and/or a saltthereof.

The present invention also provides a method of producing a feedstuffsadditive, comprising:

producing D-pantothenic acid and/or a salt thereof as described above,and

combining the D-pantothenic acid and/or a salt thereof with a carriersuitable for use in feedstuffs.

The present invention also provides a vector suitable for expressing theglyA gene from E. coli which contains a promoter and the gene sequence.

The present invention also provides a microorganism from theEnterobacteriaceae family, transformed with the vector described above.

The present invention also provides a method for producing D-pantothenicacid and/or a salt thereof by fermenting the microorganism describedabove.

In particular, yhe present invention also provides a method forproducing an animal feedstuffs additive, comprising:

(a) producing D-pantothenic acid or a salt thereof as described above,wherein the alkaline earth metal of the alkaline earth salt is magnesiumand/or calcium,

(b) optionally, removing water from the medium,

(c) separating the biomass formed during the fermentation in an amountof 0 to 100%,

(d) optionally, adding one or more magnesium and/or calcium salts ofD-pantothenic acid to the fermentation broths from (b), and

(e) producing the feedstuffs additive,

wherein the amount of the added one or more magnesium and/or calciumsalts of D-pantothenic acid is such that the amount thereof in thefeedstuffs additive is in the range from 1 about 20 to 80 wt. % based onthe dry mass of the feedstuffs additive.

BRIEF DESCRIPTION OF THE FIGURES

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1: Map of the plasmid pTrc99AglyA containing the glyA gene.

FIG. 2: Map of the plasmid pACYC184panBC containing the panBC gene.

Data relating to lengths are given as approximate values. Theabbreviations and names used are as follows:

Amp: Ampicillin resistance gene

Tc: Tetracyclin resistance gene

lacI: Gene for repressor protein of the trc promoter

Ptrc: trc promoter region, IPTG inducible

glyA: Coding region of the glyA gene

5S: 5S rRNA region

rrnBT: rRNA terminator region

panB: Coding region of the panB gene

panC: Coding region of the panC gene

The abbreviations for the restriction enzymes are as follows:

BamHI: Restriction endonuclease from Bacillus amyloliquefaciens

BglII: Restriction endonuclease from Bacillus globigii

ClaI: Restriction endonuclease from Caryphanon latum

EcoRI: Restriction endonuclease from Escherichia coli

EcoRV: Restriction endonuclease from Escherichia coli

HindIII: Restriction endonuclease from Haemophilus influenzae

KpnI: Restriction endonuclease from Klebsiella pneumoniae

PstI: Restriction endonuclease from Providencia stuartii

PvuI: Restriction endonuclease from Proteus vulgaris

SacI: Restriction endonuclease from Streptomyces achromogenes

SalI: Restriction endonuclease from Streptomyces albus

SmaI: Restriction endonuclease from Serratia marcescens

XbaI: Restriction endonuclease from Xanthomonas badrii

XhoI: Restriction endonuclease from Xanthomonas holcicola.

DETAILED DESCRIPTION OF THE INVENTION

Whenever D-pantothenic acid, pantothenic acid or pantothenate arementioned in the following, these are intended to mean not only the freeacids but also the salts of D-pantothenic acid such as e.g. the calcium,sodium, ammonium or potassium salt.

In this connection, the expression “enhancement” describes the increasein cellular activity of one or more enzymes or proteins in amicroorganism which are coded by the corresponding DNA by, for example,increasing the copy number of the gene or genes, using a strong promoteror a gene or allele which codes for a corresponding enzyme or proteinwith high activity and optionally combining these steps.

As a result of the enhancement step, in particular overexpression, theactivity or concentration of the corresponding protein is generallyincreased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%or 500%, at most up to 1000% or 2000%, with respect to that of the wildtype protein or the protein in the initial microorganism.

Microorganisms which are provided by the present invention can produceD-pantothenic acid from glucose, saccharose, lactose, fructose, maltose,molasses, starch, cellulose or from glycerine and ethanol. They aremembers of the Enterobacteriaceae family, in particular from the genusEscherichia. From the genus Escherichia, the species Escherichia coli ismentioned in particular. Within the species Escherichia coli, theso-called K-12 strains such as e.g. the strain MG1655 or W3110 (Neidhardet al.: Escherichia coli and Salmonella. Cellular and Molecular Biology(ASM Press, Washington D.C.)) or the Escherichia coli wild type strainIF03547 (Institut für Fermentation, Osaka, Japan) and mutants derivedtherefrom are suitable, these having the ability to produceD-pantothenic acid.

Suitable D-pantothenic acid-producing strains from the genusEscherichia, in particular from the species Escherichia coli are forexample

Escherichia coli FV5069/pFV31

Escherichia coli FV5069/pFV202

Escherichia coli FE6/pFE80 and

Escherichia coli KE3.

It was found that Enterobacteriaceae, after overexpression of the glyAgene coding for serine hydroxymethyl transferase produce D-pantothenicacid in an improved way.

The nucleotide sequences in the glyA gene from Escherichia coli werepublished by Plamann et al (Nucleic Acids Research11(7):2065-2075(1983)) and can also be obtained from the genome sequencefor Escherichia coli, under Accession Number AE000374, published byBlattner et al. (Science 277, 1453-1462 (1997).

The glyA gene described in the literature references cited above can beused in accordance with the invention. Furthermore, alleles of the glyAgene which are produced by degeneracy of the genetic code or byfunctionally neutral sense mutations may be used.

In order to produce an overexpression, the copy number of thecorresponding genes can be increased or the promoter and regulationregion or the ribosome bonding site which is located upstream of thestructure gene can be mutated. Expression cassettes which areincorporated upstream of the structure gene act in the same way. It isalso possible to increase expression during the course of fermentativeD-pantothenic acid production by means of inducible promoters.Expression is also improved by steps to extend the lifetime of m-RNA.Furthermore, by inhibiting degradation of the enzyme protein, enzymeactivity is also enhanced. The genes or gene structures may either bepresent in plasmids with different copy numbers or integrated andamplified in the chromosome. Alternatively, overexpression of the genesinvolved can be achieved by changing the composition of the medium andby culture management.

One skilled in the art will find instructions for this, inter alia, inChang and Cohen (Journal of Bacteriology 134:1141-1156 (1978)), inHartley and Gregori (Gene 13:347-353 (1981)), in Amann and Brosius (Gene40:183-190 (1985)), in de Broer et al. (Proceedings of the National ofSciences of the United States of America 80:21-25 (1983)), in LaVallieet al. (BIO/TECHNOLOGY 11, 187-193 (1993)), in PCT/US97/13359, in Llosaet al. (Plasmid 26:222-224 (1991)), in Quandt and Klipp (Gene 80:161-169(1989)), in Hamilton (Journal of Bacteriology 171:4617-4622 (1989), inJensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)and in well-known textbooks on genetics and molecular biology.

Plasmid vectors which are replicable in Enterobacteriaceae such as e.g.cloning vectors derived from pACYC184 (Bartolomé et al.; Gene 102, 75-78(1991)), pTrc99A (Amann et al.; Gene 69:301-315 (1988)) or pSC 101derivatives (Vocke and Bastia, Proceedings of the National Academy ofScience USA 80 (21):6557-6561 (1983)) can be used. In one processaccording to the invention, a strain transformed with a plasmid vectorcan be used, wherein the plasmid vector contains at least the nucleotidesequence coding for the glyA gene.

Furthermore, it may be advantageous for the production of D-pantothenicacid using strains from the Enterobacteriaceae family, in addition toenhancing the glyA gene, to enhance, in particular to overexpress,separately or together one or more endogenous genes selected from thegroup

the ilvGM operon coding for acetohydroxy acid synthase II (WO 97/10340)

the panB gene coding for ketopantoate-hydroxymethyl transferase (U.S.Pat. No. 5,518,906),

the pane gene coding for ketopantoate reductase (EP-A-1001027)

the panD gene coding for aspartate decarboxylase (U.S. Pat. No.5,518,906),

the panC gene coding for pantothenate synthetase (U.S. Pat. No.5,518,906),

the serC gene coding for phosphoserine transaminase (Duncan und Coggins,Biochemical Journal 234:49-57 (1986)) and

the gcvT, gcvH and gcvP genes coding for the glycine cleavage system(Okamura-Ikeda et al., European Journal of Biochemistry 216, 539-548(1993)).

Finally, it may be advantageous for the production of D-pantothenic acidusing strains of the Enterobacteriaceae family, in addition to enhancingthe glyA gene, to attenuate, in particular to switch off or express at alower level the following gene,

the avtA gene coding for transaminase C (EP-A-1001027).

The expression “attenuation” in this connection describes the reductionin, or switching off, of the intracellular activity of one or moreenzymes (proteins) in a microorganism which are coded by thecorresponding DNA by, for example, using a weak promoter or using a geneor allele which codes for a corresponding enzyme (protein) with a loweractivity or inactivates the corresponding gene or enzyme (protein) andoptionally combining these steps.

The activity or concentration of the corresponding protein is generallylowered to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of theactivity or concentration of the wild type protein or of the activity orconcentration of the protein in the initial microorganism by theattenuation step.

Furthermore, it may be advantageous for the production of D-pantothenicacid, in addition to overexpressing the glyA gene, to switch offundesired secondary reactions (Nakayama: “Breeding of Amino AcidProducing Microorganisms”, in: Overproduction of Microbial Products,Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982). Inthe process according to the invention, bacteria may be used in whichthe metabolic pathways which reduce the formation of D-pantothenic acidare at least partially switched off.

Microorganisms prepared according to the invention can be cultivated ina batch process, in a fed batch process or in a repeated fed batchprocess. Summaries of known cultivation methods are described in thetextbook by Chmiel (Bioprozesstechnik 1. Einführung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in thetextbook by Storhas (Bioreaktoren und periphere Einrichtungen (ViewegVerlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used has to satisfy the requirements of theparticular strain in an appropriate manner. Descriptions of culturemedia for various microorganisms are given in the manual “Manual ofMethods for General Bacteriology” by the American Society forBacteriology (Washington D.C., USA, 1981). Sources of carbon which areused are sugar and carbohydrates such as e.g. glucose, saccharose,lactose, fructose, maltose, molasses, starch and cellulose, oils andfats such as e.g. soy oil, sunflower oil, groundnut oil and coconutbutter, fatty acids such as e.g. palmitic acid, stearic acid andlinoleic acid, alcohols such as e.g. glycerine and ethanol and organicacids such as e.g. acetic acid. These substances may be usedindividually or as a mixture.

Sources of nitrogen which are used may be organic nitrogen-containingcompounds such as peptones, yeast extract, meat extract, malt extract,maize steep liquor, soy bean flour and urea or inorganic compounds suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The sources of nitrogen may be usedindividually or as a mixture.

Sources of phosphorus which may be used are phosphoric acid, potassiumdihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts. Furthermore, the culture mediummust contain salts of metals, such as e.g. magnesium sulfate or ironsulfate, which are required for growth. Finally, essential growthsubstances such as amino acids and vitamins can be used in addition tothe substances mentioned above. Moreover, precursors of D-pantothenicacid such as aspartate, b-alanine, ketoisovalerate, ketopantoic acid orpantoic acid and optionally their salts can be added to the culturemedium. The feedstocks mentioned can be added to the culture in the formof a one-off batch or be fed to the culture medium in an appropriatemanner during cultivation.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammoniaor ammoniacal water or acid compounds such as phosphoric acid orsulfuric acid are used in a suitable way to control the pH of theculture.

It is also possible, to prepare the alkaline earth salts of pantothenicacid, in particular the calcium salt, to add a suspension or solution ofan alkaline earth-containing inorganic compound, such as for examplecalcium hydroxide, or an organic compound such as the alkaline earthsalt of an organic acid, for example calcium acetate, continuously orbatchwise during fermentation. The cation required to prepare thedesired alkaline earth salt of D-pantothenic acid is introduced directlyinto the fermentation broth in the desired amount in this way, generallyin the ratio of 0.8 to 1.2 to 1, with respect to the pantothenic acid,preferably in stoichiometric amounts.

Atifoaming agents such as e.g. polyglycol esters of fatty acids are usedto regulate the production of foam. To maintain the stability ofplasmids, suitable selectively acting substances, e.g. antibiotics, maybe added to the medium. In order to maintain the presence of aerobicconditions, oxygen or oxygen-containing gas mixtures such as e.g. airare introduced into the culture. The temperature of the culture isusually 25° C. to 45° C. and preferably 30° C. to 40° C. The culture iscontinued until a maximum of D-pantothenic has formed. This objective isusually achieved within 10 hours to 160 hours.

The D-pantothenic acid or the corresponding salts of D-pantothenic acidcontained in the fermentation broth may then be isolated and purifiedusing known procedures. It is also possible preferably first to partly(³0 to 100%) or completely remove the biomass from the fermentationbroth containing D-pantothenic acid and/or its salts by known methods ofseparation such as, for example, centrifuging, filtering, decanting or acombination of these. However, it is also possible to leave all of thebiomass in the fermentation broth. In general, the suspension orsolution is preferably concentrated and worked up to produce a powder,for example using a spray dryer or a freeze drying unit. Then thispowder is converted into a coarse-grained, very free-flowing, storableand largely dust-free product with a particle size distribution of 20 to2000 μm, in particular 100 to 140 μm, using suitable compacting orgranulating methods, e.g. also pelletizing. The use of conventionalorganic or inorganic auxiliary substances, or supports such as starch,gelatin, cellulose derivatives or similar substances, such as areconventionally used in foodstuffs or animal feed processing as -binders,gelling agents or thickeners, or other substances such as, for example,silicas, silicates or stearates is advantageous when granulating orcompacting.

Alternatively, the fermentation product, with or without otherconventional constituents from the fermentation broth, can be depositedonto an organic or inorganic support substance which is known andconventionally used in the foodstuffs processing sector such as, forexample, silicas, silicates, grist, bran, flour, starch, sugar or othersand/or stabilized with conventional thickeners or binders. Examples ofapplications and processes for this are described in the literature (DieMühle+Mischfuttertechnik 132 (1995) 49, page 817).

Optionally, in a suitable process step, D-pantothenic acid or thedesired salt of D-pantothenic acid or a preparation containing thesecompounds is added to the product in order to produce or adjust to thedesired concentration of pantothenic acid or the desired salt.

The desired concentration is generally in the range 20 to 80 wt. % (dryweight). This range includes all specific values and subrangestherebetween, such as 30, 40, 50, 60, and 70 wt. %.

The concentration of pantothenic acid can be determined using knownchemical (Velisek; Chromatographic Science 60, 515-560 (1992)) ormicrobiological methods such as e.g. the Lactobacillus plantarum test(DIFCO MANUAL, 10th edition, p. 1100-1102; Michigan, USA).

A pure culture of the following microorganism was deposited at theGerman Collection of Microorganisms and Cell Cultures (DSMZ,Braunschweig, Germany) on 8th September 2000 in accordance with theBudapest treaty:

Escherichia coli K12 strain FE6-1, as DSM 13721.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

The minimal (M9) and complete (LB) media for Escherichia coli aredescribed by J. H. Miller (A short course in bacterial genetics (1992),Cold Spring Harbor Laboratory Press). The isolation of plasmid DNA fromEscherichia coli and all the techniques for restriction, Klenow andalkaline phosphatase treatment are performed in accordance with Sambrooket al. (Molecular cloning—A laboratory manual (1989) Cold Spring HarborLaboratory Press). The transformation of Escherichia coli, if notdescribed differently, is performed in accordance with Chung et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica USA (1989) 86: 2172-2175).

Example 1 Construction of the Expression Plasmid pTrc99AglyA

The glyA gene from E. coli K12 is amplified using the polymerase chainreaction (PCR) and synthetic oligonucleotides. Starting from thenucleotide sequence for the glyA gene in E. coli K12 MG1655 (AccessionNumber AE000341, Blattner et al. (Science 277, 1453-1462 (1997),) PCRprimers are synthesized (MWG Biotech, Ebersberg, Germany):

glyA1: 5′-GTTAGCTGAGTCAGGAGATG-3′

glyA2: 5′-TACGCTTATCAGGCCTACAC-3′

The chromosomal E. coli K12 MG1655 DNA used for the PCR is isolatedaccording to data provided by the manufacturer using “QiagenGenomic-tips 100/G” (QIAGEN, Hilden, Germany). An approximately 1400 bpsize DNA fragment can be amplified with specific primers under standardPCR conditions (Innis et al. (1990) PCR Protocols. A Guide to Methodsand Applications, Academic Press) using Pfu DNA polymerase (PromegaCorporation, Madison, USA). The PCR product is ligated according to dataprovided by the manufacturer using the vector pCR-Blunt II-TOPO (ZeroBlunt TOPO PCR Cloning Kit, Invitrogen, Groningen, Netherlands) andtransformed in E. coli strain TOP10. The selection of cells carrying theplasmid is performed on LB agar which has been treated with 50 μg/mlkanamycin. After isolation of the plasmid DNA, the vector pCR-BluntII-TOPOglyA is cleaved with restriction enzymes HindIII and XbaI and theglyA fragment is isolated after separation in 0.8% agarose gel using theQIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). The vectorpTrc99A (Pharmacia Biotech, Uppsala, Sweden) is cleaved with the enzymesHindIII and XbaI and ligated with the isolated glyA fragment. E. colistrain XL1-Blue MRF′ (Stratagene, La Jolla, USA) is transformed with theligation mixture and cells carrying the plasmid are selected on LB agarwhich has been treated with 50 μg/ml ampicillin. Successful cloning canbe detected after plasmid DNA isolation by control cleavage with theenzyme SspI. The plasmid is called pTrc99AglyA (FIG. 1).

Example 2 Preparing the Strain FE6-1/pTrc99AglyA

E. coli strain FE6 is a valine-resistant mutant of E. coli K12 MG1655(U.S. Pat. No. 6,171,845) and is deposited as DSM12379 at the GermanCollection of Microorganisms and Cell Cultures (DSMZ, Braunschweig,Germany). Starting from FE6, spontaneous mutants are isolated afterincubation at 37° C. on minimal agar which has been treated with 2 g/lglucose and 1 g/l β-hydroxyaspartic acid. A selected β-hydroxyasparticacid-resistant single colony is then incubated at 37° C. on minimal agarwhich contains 2 g/l glucose and 0.2 g/l O-methylthreonine. A mutantcalled FE6-1 is resistant to valine, a-ketoisovaleric acid,β-hydroxyaspartic acid and O-methylthreonine following this step. Theplasmid pTrc99AglyA is transformed in strain FE6-1 and cells carryingthe plasmid are selected on LB agar which has been treated with 50 μ/mlampicillin. The strain obtained is called FE6-1/pTrc99AglyA.

Example 3 Preparing the Strain FE6-1/pTrc99AglyA,pACYC 184panBC

The D-pantothenic acid-producing E. coli strain FV5069/pFV31 isdescribed in EP-A-0590857 and is deposited as FERM BP 4395 in accordancewith the Budapest treaty. The plasmid pFV31 is isolated fromFV5069/pFV31 and cleaved with restriction enzyme EcoRI. After separationin 0.8% agarose gel, the approximately 2600 bp size DNA fragment onwhich the panBC genes are present is isolated using the QIAquick GelExtraction Kit (QIAGEN, Hilden, Germany). The vector pACYC184 (Chang, A.C. Y. und Cohen, S. N., Journal of Bacteriology 134, 1141-1156 (1978);ATCC37033 (American Type Culture Collection, Manassas, USA)) is cleavedwith the enzyme EcoRI and ligated with the isolated panBC fragment. E.coli strain FE6-1 is transformed with the ligation mixture and cellswhich carry the plasmid are selected on LB agar which has been treatedwith 10 μg/ml tetracyclin. Successful cloning can be detected by controlcleavage with the enzymes EcoRV and EcoRI after plasmid DNA isolation.The plasmid is called pACYC184panBC (FIG. 2). The strainFE6-1/pTrc99AglyA described in example 2 is transformed with the plasmidpACYC184panBC. Selection is performed on LB agar which has been treatedwith 50 μg/ml ampicillin and 10 μg/ml tetracyclin. The strain producedin this way is called FE6-1/pTrc99AglyA, pACYC184panBC.

Example 4 Production of D-Pantothenic Acid with Strains Derived fromFE6-1

Pantothenate production by E. coli strains FE6-1, FE6-1/pTrc99AglyA,FE6-1/pACYC184panBC, FE6-1/pTrc99AglyA, pACYC184panBC is checked inbatch cultures of 10 ml which are contained in 100 ml conical flasks.For this, 10 ml of preculture medium with the following composition: 2g/l yeast extract, 10 g/l (NH₄)2SO₄, 1 g/l KH2PO4, 0.5 g/l MgSO₄·₇H₂O,15 g/l CaCO₃, 20 g/l glucose, is inoculated with a single colony andincubated for 20 hours at 33° C. and 200 rpm in an ESR incubator fromKühner AG (Birsfelden, Switzerland). 200 μl portions of this precultureare each inoculated into 10 ml of production medium (25 g/l (NH₄)₂SO₄, 2g/l KH₂PO₄, 1 g/l MgSO₄·₇H₂O, 0.03 g/l FeSO₄·₇H₂O, 0.018 g/l MnSO₄·₁H₂O,30 g/l CaCO₃, 20 g/l glucose, 20 g/l β-alanine, 250 mg/l thiamine) andincubated for 48 hours at 37° C. During the incubation ofFE6-1/pTrc99AglyA, 50 mg/l ampicillin are also added to the media,during the incubation of FE6-1/pACYC184panBC, 10 mg/l tetracyclin arealso added to the media and during the incubation ofFE6-1/pTrc99AglyA,pACYC184panBC, 50 mg/l ampicillin and 10 mg/ltetracyclin are also added to the media. After incubation, the opticaldensity (OD) of the culture suspension is determined at a testwavelength of 660 nm using a LP2W photometer from the Dr. Lange Co.(Düsseldorf, Germany).

Then the concentration of D-pantothenate formed in the sterile filteredculture supernatant liquid is determined using the Lactobacillusplantarum ATCC8014 pantothenate assays in accordance with data fromDIFCO (DIFCO MANUAL, 10th Edition, p. 1100-1102; Michigan, USA). Thecalcium salt of D(+)-pantothenic acid hydrate (catalogue number25,972-1, Sigma-Aldrich, Deisenhofen, Germany) is used for calibrationpurposes.

Table 1 gives the results of the test.

TABLE 1 OD Pantothenate Strain (660 nm) mg/l FE6-1 10.6 19FE6-1/pTrc99AglyA 9.2 39 FE6- 8.4 540 1/pACYC184panBC FE6-1/pTrc99AglyA,9.4 820 pACYC184panBC

The publications cited above are incorporated herein by reference.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

This application is based on German Patent Application Serial No. 101 06461.6, filed on Feb. 13, 2001, which is incorporated herein byreference.

What is claimed is:
 1. A method of producing D-pantothenic acid and/or asalt thereof, comprising: fermenting a microorganism of the familyEnterobacteriaceae, in which the nucleotide sequence for the endogenousglyA gene is overexpressed in a medium suitable for the production ofserine hydroxymethyl transferase, wherein the microorganism isEscherichia coli and produces the D-pantothenic acid and/or a saltthereof.
 2. The method of claim 1, wherein the medium is enriched in theD-pantothenic acid and/or a salt thereof and/or the cells of themicroorganism are enriched in the D-pantothenic acid and/or a saltthereof.
 3. The method of claim 1, further comprising isolating at leasta portion of the D-pantothenic acid and/or a salt thereof from themedium.
 4. The method of claim 1, further comprising isolating at leasta portion of the biomass from the medium.
 5. The method of claim 1,wherein the fermentation is performed in the presence of at least onealkaline earth salt, which is supplied continuously or batchwise to themedium, and a product containing an alkaline earth salt of D-pantothenicacid is produced.
 6. The method of claim 1, wherein one or more of theendogenous genes selected from the group consisting of the ilvGM operoncoding for acetohydroxy acid synthase II, the panB gene coding forketopantoate hydroxymethyl transferase, the panE gene coding forketopantoate reductase, the panD gene coding for aspartatedecarboxylase, and the panC gene coding for pantothenate synthetase areoverexpressed in the microorganism.
 7. The method of claim 1, whereinthe activity or concentration of the glyA gene product is increased by10 to 2000%, with respect to that of the protein in the initialmicroogranism.
 8. The method of claim 1, wherein transformed strains areused in which the glyA gene is present in plasmids or is integrated intothe chromosome.
 9. A method for producing D-pantothenic acid and/or asalt thereof by fermenting a transformed microorganism wherein themicroorganism is Escherichia coli transformed with a vector containing apromotor operably linked to the Escherichia coli glyA gene.
 10. A methodfor producing D-pantothenic acid and/or a salt thereof by fermenting atransformed microorganism wherein the microorganism is Escherichia colitransformed with pTrc99AglyA.